This application, Neuro-glio-vascular interactions in vivo probed with optical imaging, will address the broad challenge of the BRAIN Initiative (Innovative Neurotechnologies) and the targeted challenge (tools to target, identify and characterize non-neuronal cells in the brain). The central goal of this proposal is to develop a novel multimodality optical tool aimed at enabling concomitant cell-specific imaging of synchronized astrocytic and neuronal activities and local neurovascular network interactions at cellular and capillary resolution level. Emphasis is on developing tools that allow complete characterization of the cortical vascular tree at high temporal resolution and use this combined with genetically encoded Ca2+ fluorescence indicators to probe the role of astrocytes in neurovascular regulation. Specifically, this proposal seeks development of a multimodality fluorescence ? swept-source optical Doppler microscopy (fl-ssODM). This approach enables imaging of large-scale astrocytic/neuronal Ca2+ fluorescence and 3D cerebral blood flow velocity (CBFv) networks for real-time concomitant detection of neuronal-glio-vascular activities in mouse cortex in vivo (Aim 1, Aim 2). The proposed ssODM is stimulated by our recent breakthrough in swept-source ODM that facilitates, for the first time, 3D imaging of the entire vascular network and quantitation of CBFv in capillaries in all layers of mouse cortex (>1.8mm). The innovative differential Doppler phase detection (Aim 2) will further increase volumetric rate over 40 times (e.g., to 20 volumes/s) to enable imaging dynamic changes in CBFv and vasodilation/constriction at capillary resolution. In combination with viral delivery of genetically encoded Ca2+ indicators, e.g., GCaMP6f for astrocytes and jRGECO1a for neurons in GFAP-cre mice, fl-ssODM will allow us to observe the spatiotemporal evolution of astrocyte/neuronal transient activities and the concomitant vascular responses at rest and during activation of the cortex. To validate fl-ssODM for studying the roles of astrocytes in neuro-glio-vascular interactions, we propose to characterize the changes in astrocytic/ neuronal Ca2+ signaling and vascular response to brain stimulation after inhibition of astrocyte signaling using designer receptor exclusively activated by designer drugs (DREADDs) (Aim 3). An interdisciplinary research team has been assembled, including biomedical engineers, neuroimaging scientists, and neuroscientists to conduct the proposed research and development. The broad impact of this application will be to provide a tool to investigate the interactions between astrocyte, neuronal activities and regional CBFv responses in the brain.